EFFECTS OF TEMPERATURE AND MEDIA ON
PRESERVATION AND RECOVERY OF Mycobacterium
tuberculosis STRAINS
FRED ONGERA ORINA
MASTER OF SCIENCE
(Mycobacteriology)
2009
1
Effects of temperature and media on preservation and recovery of
Mycobacterium tuberculosis strains
FRED ONGERA ORINA
A thesis submitted in partial fulfillment for the Degree Master of
Science in Mycobacteriology in the
Jomo Kenyatta University of Agriculture and Technology
2009
i
DECLARATION
This is my original work and has not been presented for a degree in any other
university.
Signature………………………………
Date………………………………
Fred Ongera Orina
This thesis has been submitted for examination with our approval as the
university supervisors.
1. Signature……………………..……
Date…………………………….
Dr. Willie Githui
KEMRI, Kenya.
2. Signature…………….………..……
Date……….……….…………..
Dr. Hellen Meme
KEMRI, Kenya.
3. Signature……………………………
Date…………………………..
Prof. Rosebella Maranga
JKUAT, Kenya.
ii
DEDICATION
I wish to dedicate this work to my mentor Dr. W. Githui and my mother Juliana; both
of you have been patient with me. Thank you
iii
ACKNOWLEDGMENTS
I thank God for his blessings to accomplish this work.
My sincere gratitude to my supervisors, Dr.Willie Githui, Dr. Helen Meme
and Prof. Rosebella Maranga for their dedicated guidance through out the
study.
I wish to thank the director Center for Respiratory Diseases Research and the
ITROMID fraternity for their contribution to the study.
I also wish to appreciate the dedicated technical assistance by Mr. Earnest
Juma, Ms. Phoebe Mumbi, Mr. Francis Karimi, Michael Musili and Mr. Peter
Kinyanjui.
My appreciation to my family for the encouragement during this period of
study and their love and support and especially Jossy kwamboka for moral
support.
My sincere gratitude to my statisticians Mr. Moses Mwangi and Mr. Jeff
Okallo for their good work
.
iv
TABLE OF CONTENTS
DECLARATION……………………………………………………………
……...i
DEDICATION………………………………...…………………………………
…ii
AKNOWLEDGEMENTS………..…………….…...…………………………….
..iii
TABLE
OF
CONTENTS………….…………...………………………………..... iv
LIST
OF
TABLES………………….………...…………………………………...viii
LIST
OF
FIGURES……………….…………...……………………………….…..ix
LIST OF ABBREVIATIONS………….……………………………………….....xi
ABSTRACT………………………………………………………………………..
xiii
CHAPTER
ONE…………………………….………………………….….………1
1.0
INTRODUCTION...................................................................................................1
v
1.2 STATEMENT
OF
THE
PROBLEM…………………………………………....3
1.3 JUSTIFICATION…..….…..……………...………………………………...
…..4
1.4 HYPOTHESIS……………………………...………….……………….…….
…5
1.4.1
Null
hypothesis
(HO)….………………………..……...….………5
1.4.2
Alternative
hypothesis
(Ha)
……….….…………….….…………5
1.5 OBJECTIVES……………………...……………..…………………………
…..6
1.5.1
Main
objective……....….………………..………..………………6
1.5.2
Specific
objectives………………………………………...……….6
CHAPTER
TWO………………………………………...………………………..7
2.0 Literature
review……………………………………………………….……….7
2.1Classification
of
mycobacteria………………………………………..………....7
vi
2.2
Genome
structure
of
mycobacteria……….……………………………...….7
2.3
Mycobacterial
cell
structure
and
metabolism……………….…………...….8
2.4
Mycobacterium
tuberculosis
(MTB)……………………….………….….....8
2.4.1
General
characteristics
of
MTB……..……….………….…....8
2.4.2
Cell
wall
structure
of
MTB…………………….………….…...9
2.4.3
Virulence
mechanism
and
factors
of
M.
tuberculosis………..11
2.4.4
Pathogenesis
of
MTB………………….….…….………....…13
2.4.5
Transmission
of
tuberculosis
…………………….…….……..14
2.4.6
Diagnosis
of
tuberculosis……………………………....……..14
2.4.7
Tuberculosis
treatment……………………..…….….………15
2.4.8
Drug
resistance
tuberculosis………………….…….……...15
vii
2.5
Preservation of microorganisms……………………….…..…………16
2.5.1
Methods
of
preservation
of
microorganisms………….………16
2.5.2
Effects
of
freezing
on
microorganisms…….…….…………..18
2.5.3
Effects
of
thawing
on
microorganisms…………………….…20
2.6
medium
used
in
preservation……………………………………………...21
2.6.1
Gelatin………………………………….………………..21
2.6.2
Monosodium
glutamate
(MSG)…………………
….……….22
2.6.3
Trypticase soy broth with 20% glycerol…………...……23
2.6.4
Glycerol……………………………………….…………23
2.6.5
Sterilized distilled water….……………….……...…23
2.6.6
Phosphate buffer saline (PBS)………… ……..………...24
2.6.7
Lactose…………………………………….………...…….…
.25
CHAPTER
THREE……………………………..………………………………..29
3.0 Methodology……………………………………………..….…….……..…
….29
viii
3.1 Study
design………………………………………………….….……….….…29
3.2 Study
site…………………………………………………….….……….……..29
3.3 Study
population,
inclusion
and
exclusion
criteria……………….……………29
3.4 Sampling……..…………………………………………….…………….…
….29
3.5 Preparation
of
storage
media…………………………….……..……………...30
3.5.1
Trypticase
soy
broth
20%
glycerol…………………...…….….30
3.5.2
OADC
enriched
middlebrook
7h9
broth
+
5%
glycerol……...30
3.5.3
Middlebrook
7H9
broth……………………………..………...30
3.5.4
Lactose
broth
……………………..……………….………….30
3.5.5
Phosphate
buffered
saline…………………………..…………31
3.5.6
Skim
milk
medium……………….……………………………31
ix
3.5.7
Sodium
glutamate
medium……………………...…………….31
3.5.8
Lowenstein
Jensen
medium…………………..……..………...32
3.6 Subculture
of
stored
strains………………………………….….…………….33
3.7 Storage
suspension……………………………………….……….……..……33
3.8 Freezing and thawing of MTB strains.....…………………………..………
...33
3.8.1
Approach
one
…………...………………………………..……33
3.8.2
Approach
two……………...………….…..….…….…………34
3.9 Inoculation
of
preserved
suspension…………...….…………………………...34
3.10
Reading
of
cultures………………….………...…………………….…….34
3.11
Statistical
analysis………………….……………….……………………..35
CHAPTER
FOUR………………………………………………………..….……36
x
4.0
RESULTS…………………..……………………..……….…………………..36
CHAPTER
FIVE…………………………………………………………………60
5.0
DISCUSSION………...…………………………….…...…………………..…60
CHAPTER
SIX………………………………………………………………...…71
6.1 CONCLUSION………………………………..….……...………………….
…71
6.2 RECOMMENDATIONS
………………...………………………….………...72
7.0
REFERENCE…………………………….....…..……………...………………73
APPENDICES………….…….………………….….….…………..……….……
..84
xi
LIST OF TABLES
Table 1:
Preparation
of
mineral
of
culture
salts……...………………………………..88
Table 2:
Reporting
results………………………………………..90
Table 3:
Equipment
for
preservation…….……..……………………………92
LIST OF FIGURES
Figure 1:
Schematic
representation
of
MTB
cell
wall
structure…………….11
Figure 2:
Molecular
structure
of
gelatin…………………………………….24
xii
Figure: 3
Chemical
structure
of
MSG
…………………………..…………..25
Figure 4:
Molecular
structure
of
lactose…………………………..…………28
Figure 5:
Monthly MTB growth recovery on 5% sodium glutamate.
….……37
Figure 6:
Monthly
MTB
growth
recovery
on
skimmed
milk...........................39
Figure 7:
Monthly
MTB
growth
recovery
on
7H9
OADC…………………...41
Figure 8:
Monthly
MTB
growth
recovery
on
Middlebrook
7H9……..……...43
Figure 9:
Monthly
MTB
growth
recovery
on
recovery
on
recovery
on
recovery
on
lactose………………………...45
Figure 10:
Monthly
MTB
growth
gelatin…….…………………..47
Figure 11:
Monthly
MTB
growth
PBS……….…………….……49
Figure 12:
Monthly
MTB
growth
TSB……………………….….51
Figure 13:
Monthly
growth
recovery
for
MTB
on
SDW…………………..….52
xiii
Figure 14:
Growth
grading
concordance
for
media
at
-
for
media
at
-
80ºC……………….…53
Figure 15:
Growth
grading
concordance
20ºC…….……..…….54
Figure16:
Growth
grading
concordance
for
media
at
4ºC………………..…..55
Figure 17:
Growth grading concordance for media at room temperature
...…..56
Figure 18:
Direct freezing and direct thawing in recovery of strains
………....58
Figure 19
Correlation of recovery between approach one and approach two
in
recovery
of
MTB…………………………………………………...59
xiv
LIST OF APENDICES
Apendix1
SOP Preparation
media sedfor preservation…………………………...84
Apendix2
SOP Preparation media for subculture of
MTBstrains… …………...…..88
Apendix3
Reportingofsubcultureresults………………………….……………….90
Apendix4
Quality
control…………………………………………..………………91
xv
LIST OF ABBREVIATIONS
AFB
Acid-fast bacilli
AIDS
Acquired immunodeficiency syndrome
CMI
Cell mediated immunity
CRDR
Centre for Respiratory Diseases Research
DTH
Delayed type hypersensitivity
EMB
Ethambutol
Gelatin medium
1% gelatin buffered at pH 6.8;
HIV
Human immunodeficiency virus
INH
Isoniazid
Initial
Subcultures reading immediately on day zero of preservation
ITROMID
Institute of Tropical Medicine and Infectious Diseases
JKUAT
Jomo Kenyatta University of Agriculture and Technology
KEMRI
Kenya Medical Research Institute
Lactose medium
15% aqueous solution of lactose at pH 5.0
LJ
Lowenstein Jensen medium with glycerol
MDR-TB
Multi Drug Resistant Tuberculosis
MP
Mononuclear phagocytes
MSG
Monosodium glutamate
MTB
Mycobacterium tuberculosis
NTM
Non tuberculous mycobacteria
OADC
Oleic acid , Albumin, Dextrose, Catalase
xvi
PBS medium
Phosphate buffer saline pH 7.2
PMN
Polymorphonuclear neutrophills
PZA
Pyrazinamide
RIF
Rifampin
RT
Room temperature (25ºC)
SDW
Sterilized distilled water
SG medium
5% sodium glutamate + glycerol
SM medium
Skimmed milk
SOP
standard operating procedures
STREP
Streptomycin
τ
Kendall’s coefficient of concordance
TB
Tuberculosis
TSB medium
Trypticase Soy Broth with 20% Glycerol
WHO
World health organization
ZN
Ziehl Neelsen
7H9 OADC medium OADC-enriched Middle brook 7H-9 medium with 5%glycerol
7H-9 medium
Middle brook 7H-9
xvii
ABSTRACT
The preservation and recovery of Mycobacterium tuberculosis (MTB) strains isolated
from clinical specimens is an important stage in the understanding of issues
pertaining to tuberculosis control. Well preserved strains provide readily available
and cost effective material that is useful in facilitating studies that address pertinent
issues.
This study was done to determine optimum temperature(s), suitable media and
conditions for recovery that sustain high survival rate of MTB strains for five months
at Center for Respiratory Diseases Research KEMRI laboratory.
A total of 97 randomly selected strains were aliquoted into two aliquots of the
suspension media containing each; 1% gelatin buffered pH 6.8 (gelatin), 15%
aqueous solution of lactose pH 5.0 (lactose), 5% sodium glutamate + glycerol (SG),
trypticase soy broth + glycerol (TSB), phosphate buffered saline pH 7.2 (PBS),
Middlebrook 7H9 (7H9), skimmed milk (SM), OADC-enriched Middlebrook 7H9 +
5%glycerol (OADC-7H9) and sterilized distilled water (SDW).
The aliquots were preserved using two approaches each utilizing four temperatures:
room temperature (RT), 4˚C, -20˚C, and -80˚C. In the first approach, strains were
preserved directly to the respective temperature. In the second approach a systematic
step method starting from RT to 4˚C, to -20˚C, and to -80˚C with one hour interval,
was utilized. After each subsequent month for five months, strains were thawed by
systematic approach starting from -80˚C to -20˚C to 4˚C to RT. For the second
approach strains were directly thawed after five months preservation. The thawed
xviii
strains by both approaches were subcultured on Lowenstein Jensen, incubated at 37ºC
for four weeks and -growth was graded according to the standard method.
Cross-tabulation of grading of growth was performed to determine interrelation
between temperature and media using 7H9 medium as the standard. Significant
differences within and between the grading of each suspension media were
determined by using the Kendall’s coefficient of concordance. A p<0.05 was
regarded as significant.
In the first approach at -80ºC OADC-7H9 had the highest yield while SDW had the
lowest yield (p<0.05). At -20ºC SG had highest yield while SDW had the lowest yield
(p<0.05). At 4ºC SG had highest yield while PBS had the lowest yield (p<0.05). At
RT SM had the highest yield while TSB had the lowest grading (p<0.05).
In the second approach, at -80ºC, 5% SG had the highest yield while SDW had the
lowest yield p<0.05. At -20ºC, 5% SG had highest yield while SDW had the lowest
yield p<0.05. At 4ºC, 5% SG had highest yield while PBS had the lowest yield
(p<0.05). At RT SM had the highest yield while TSB had the lowest grading
(p<0.05).
When approaches one and two were compared for temperature and media, there was
no statistically significant difference (p<0.05).
This study showed that optimum recovery of MTB strains was mainly dependant on a
combination of appropriate temperature and suitable media. The media that was
consistent with high recovery was sodium glutamate while the best preservation
temperature was -80ºC. More studies are required to determine the effect of
xix
preservation using approach one and recovery using approach two and vice versa In
addition extension of the preservation duration beyond five months should be further
elucidated.
xx
CHAPTER ONE
1.0 INTRODUCTION
A total of 2 billion people (1/3 of the world’s population) are infected with
tuberculosis (TB). Globally, 9.2 million new cases and 1.7 million deaths from TB
occurred in 2006, of which 0.7 million cases and 0.2 million deaths were in HIV coinfected persons TB being the leading cause of death in AIDS patients, the TB and
HIV form a deadly combination with each multiplying the impact of the other in
which about 200,000 people with HIV die from TB every year, most of them being in
Africa. This problem has further been magnified by development of multidrug
resistant tuberculosis (MDR-TB) whereby 450,000 new MDR-TB cases are estimated
to occur every year and further complicated by the emergence of extensively drugresistant TB (WHO, 2008).
The resurgence of TB is fuelled by the emergence of MDR. Drug resistance in TB
occurs as a result of tubercle bacillus mutations. These mutations are not dependent
upon the presence of the drug. When exposed to a single effective anti-TB
medication, the predominant bacilli sensitive to that drug are killed, while the few
drug resistant mutants likely to be present if the bacterial population is high multiply
freely (Jacobs, 1994).
Developing countries like Kenya have shown an upsurge in resistant tuberculosis
bacilli (Githui et al., 1998). With the upsurge of tuberculosis and presence of drug
resistant strains, it is important to preserve the tubercle strains especially in
1
laboratories engaged in research. Archiving provides precious strain resource that
support proficiency testing in laboratories around the world, facilitates test
development and evaluation. An obstacle to development of new diagnostic tests for
tuberculosis is due to lack of access to viable reference and test strains (Tarshis,
1961). However, various methods such as freeze drying and cryopreservation have
been used in the preservation of bacteria for long periods of time. In cryopreservation,
cells or whole tissues are preserved by cooling to sub-zero temperatures, such as 80°C or -196°C (Porubcan et al., 1975).
In freeze drying process, microorganism are desiccated while in a frozen state,
moisture is removed by sublimation and cells do not collapse since they are dried
while in frozen state (Porubcan et al., 1975). Sufficiently lowering the temperature to
sub zero enhances viability in dormant state thereby permitting preservation of the
strains for extended periods of time (Horward et al., 1967).
The preservation of M. tuberculosis complex strains isolated from clinical
specimens is important for epidemiological investigations related to tuberculosis,
evolutional studies as well as a tool in the search for new regimens. In this study the
determination of an optimum medium and temperature of preservation after five
months of preservation and subculture was done. This would enable the establishment
of a standardized basic preservation method for Mycobacterium tuberculosis (MTB),
which would foster better archiving method of representative strains of biomedical
importance and facilitate characterization of these strains with respect to taxonomic
classification, infectivity, and virulence.
2
1.2
STATEMENT OF THE PROBLEM
The emergence of multi-drug resistance tuberculosis and extensive drug
resistance tuberculosis strains of Mycobacterium tuberculosis has become a challenge
in control of tuberculosis. Due to lack of easily accessible, well characterized and
viable strains, evolutional studies of tuberculosis and research as a whole has
impaired especially in resource poor countries. Africa contributes most cases of
tuberculosis amongst the 22 high TB burdened countries. However, it has very few
laboratory facilities which isolate M. tuberculosis. Even those which do, they rarely
document methods and processes for preservation and recovery.
Freeze drying method has been used in preservation of some species of
Mycobacteria, but they have proved difficult to freeze-dry resulting in relatively poor
survival rates. Freeze drying is a challenge to resource poor countries due to; high
capital cost of equipment, high energy costs and long processing time which is
typically 24 hour drying cycle. With cost being the major impediment in developing
countries it is important to standardize an archiving process that will make available
these strains affordably.
3
1.3
JUSTIFICATION
With culture of MTB becoming routine in diagnosis of TB, currently the
methods of preservation and recovery are tedious and indicate high contamination
rates of MTB strains after subculturing. This therefore complicates research which
relies mostly on well archived and viable strains.
The establishment of a standardized basic preservation method is important to
ensure availability of representative strains of biomedical importance for
epidemiological investigations related to TB evolutional studies, providing a precious
resource that facilitates the development of diagnostics, enable proficiency testing in
laboratories and evaluation tool in the search for novel regimens and for training
purposes.
4
1.4
HYPOTHESIS
1.4.1
Null hypothesis (HO)
The viability of M. tuberculosis is not different when stored and recovered at different
temperatures and media
1.4.2
Alternative hypothesis (HA)
The viability of M. tuberculosis is variable when stored and recovered at different
temperatures and media
5
1.5
OBJECTIVES
The objectives of this study were:
1.5.1
Main objective
The main objective of the study was to determine the optimum temperature(s),
suitable media and conditions that sustained high survival rate of Mycobacterium
tuberculosis (MTB) strains archived at Center for Respiratory Diseases Research
(CRDR-KEMRI) laboratory.
1.5.2
Specific objectives
1. To determine suitability of different preservation media in sustaining viability
of M. tuberculosis strains when preserved at different temperatures.
2. To determine the viability of M. tuberculosis strains after preservation at
different temperatures on different media and specific time after sub-culture
on Lowenstein Jensen media (glycerol) media.
3. To determine if direct thawing and freezing of M. tuberculosis strains affects
the recovery of the preserved strain on different media.
4. To determine if systematic thawing and freezing affects the viability of the
preserved M. tuberculosis strain on different media.
6
CHAPTER TWO
2.0 LITERATURE REVIEW
2.1Classification of Mycobacteria
The genus Mycobacterium is scientifically classified in the phylum
Actinobacteria, order Actinomycetales, suborder Corynebacterineae, and family
Mycobacteriaceae (Lehmann and Neumann, 1896). Mycobacteria can also be
classified into two categories according to the rate of growth i.e. the fast-growing
kind, slow-growing kind, and most share some common characteristics.
There is further classification of mycobacteria into several groups including:
Mycobacterium tuberculosis (MTB) complex which can cause tuberculosis
comprising of M. tuberculosis, M. africanum, M. bovis, M. canetii, and M. microti,
which can further be divided into various subspecies. The non tuberculous
mycobacteria (NTM) are all the other mycobacteria which can cause pulmonary
disease resembling tuberculosis, lymphadenitis, skin disease, or disseminated disease
(Ratledge et al., 1982).
2.2 Genome Structure of Mycobacteria
The genomes of both MTB and M. leprae have been sequenced. The genome of
M. tuberculosis is 4,411,522 base pairs long with 3,924 predicted protein-coding
sequences, and a relatively high G+C content of 65.6% (Cole, 1998). With a 4.4 Mbp,
MTB has one of the highest known bacterial genomes, coming in just short of E. coli
and a distant third to Streptomyces coelicolor (Rama et al., 1998). In contrast to the
7
MTB, the genome of M. leprae is 3,268,203 base pairs long, with only 1,604
predicted protein-coding regions, and a G+C content of about 57.8%.
2.3 Mycobacterial cell Structure and Metabolism
As deduced from its genome, MTB has the potential to manufacture all of the
machinery necessary to synthesize all of its essential vitamins, amino acids, and
enzyme co-factors. The inability to culture M. leprae, suggests the loss of many of its
metabolic capabilities making it an obligate parasite of man and therefore dependent
on its host for most of its nutritional needs. This is in accordance to its severely
degenerated genome (Cole et al., 2001). MTB genome codes for unusual cell wall
structure, with an additional layer beyond the peptodiglycan layer, which is rich in
unusual lipids, glycolipids, and polysaccharides.
2.4Mycobacterium tuberculosis (MTB)
2.4.1
General Characteristics of MTB
Mycobacterium
tuberculosis
is
weakly
Gram-positive,
non-motile,
pleomorphic rod, measuring 2-4 μm in length and 0.2-0.5 μm in width (Minnikin,
1982). It is an obligate aerobe growing most successfully in tissues having the highest
partial pressure of oxygen such as lung apices.
It is a facultative intracellular pathogen preferentially utilizing mononuclear
phagocytes (MP) as its habitat. It can inhabit also nonprofessional phagocytes usually
the macrophages. Some mycobacteria tend to be fastidious (difficult to culture), with
some species having extremely long reproductive cycles (M. leprae, for example,
may take more than 20 days to proceed through one division cycle). Under favorable
8
laboratory conditions, M. tuberculosis divides every 12 to 24 hours. This pace is
extremely slow compared to that of most cultivable bacteria, which duplicate at
regular intervals ranging from about 15 min to one hour (Chauhan, 2006). The slow
growth rate might be partially determined by the cell wall impermeability that limits
nutrient uptake (Niederweis, 2008).
Mycobacterium tuberculosis cells are hydrophobic and tend to clump together
making them impermeable to the usual stains such as Gram's stain. Tuberculosis
complex strains are classified as acid-fast bacilli (AFB) due to their ability to retain
the Carbol- fuchsin red dye after washing with acid, alcohol, or both.
Two common solid media used for growing MTB are Middlebrook medium
which is an agar based medium and Lowenstein-Jensen medium which is an egg
based medium on which MTB colonies appear as low and buff colored when grown
on either medium. From positive sputum it takes up to 4-6 weeks to get visual
colonies on either type of media. Chains of cells in smears made from in vitro grown
colonies often form distinctive serpentine cords (Anargyros et al., 1990; Behling
1993).
As a genus, they share a characteristic cell wall, thicker than in many other
bacteria, hydrophobic, waxy and rich in mycolic acids/mycolates. Their cell wall
makes a substantial contribution to the hardiness of this genus (Brennan, 1989;
Kohsaka, 1993).
9
2.4.2
Cell Wall Structure of MTB
The cell wall structure of MTB and other mycobacteria is unique among
procaryotes and it is a major determinant of virulence for the bacterium. The most
distinctive feature of the cell wall is that up to 60% of the body weight is occupied by
lipids (mycolic acid). In addition to lipids of covalently linked skeleton, several types
of ‘extractable lipids’ including trehalose containing glycolipids, phenolic glycolipids
may be present (Brennan, 1989; Kohsaka, 1993; Niederweis, 2003).
This skeleton is composed of three covalently linked sub-structures of
peptidoglycan, arabinoglycan (AG) and mycolic acids. The peptidoglycan has two
exceptional features; the muramic acid is N-glycolylated instead of the more typical
N-acetylation and the cross links include bonds between diaminopemelic acid and Dalanine. This peptidoglycan is linked to AG via a phosphodiester bridge. The non
reducing termini of the AG polysaccharide consists of brunched penta-arabinose units
about two thirds which are esterified each with four mycolic acid residues.
In the schematic representation of mycobacterial cell wall (figure1), the
cytoplasmic membrane is encapsulated by a layer of peptidoglycan. The
peptidoglycan backbone is attached to arabinogalactan through an unusual
disaccharide phosphate linker region. The arabinogalactan is a brunched chain
polysaccharide consisting of a proximal galactose chain linked to a distal arabinose
chain. The hexaarabinofurosyl termini of arabino galactose are esterified to mycolic
acids.
10
Figure 1:
Schematic representation of MTB cell wall structure
The mycolic acid chains are shown to be perpendicular to the cytoplasmic
membrane with exposed chains of trehalose dimycolate. Another major component,
non covalently associated to the mycobacterial cell wall is immunogenic
lipoarabinomannan,
which is
attached to
the cytoplasmic membrane
by
phosphatidylinositol anchor.
These long fatty acids (C60 to C90), on the cell wall gives it a unique structure
in which the high amounts of unusual lipids covalently link to the underlying
arabinogalactan-peptidoglycan complex (Brennan and Nikaido, 1995). These
components being extremely hydrophobic, they form an exceptionally strong
permeability barrier. This renders the MTB naturally resistant to a wide variety of
11
antimicrobial agents (Jarlier
and Nikaido, 1994). Impermeability to stains and dyes;
resistance to killing by acidic and alkaline compounds; resistance to osmotic lysis via
complement deposition and resistance to lethal oxidations; survival inside the
macrophages may contribute longevity, trigger inflammatory host reactions during
pathogenesis as well as result to clumping together of MTB cells when suspended in
water (Brennan and Nikaido, 1995; Kohsaka, 1993; Minnikin, 1982).
2.4.3
Virulence Mechanisms and Virulence Factors of M. tuberculosis
Small hydrophilic molecules diffuse through water filled protein channels,
porins, whereas hydrophobic compounds use the lipid pathway (Paula et al., 1996;
Trivedi et al., 2004; Trias and Benz 1994). They are thought to be a significant
determinant of virulence in MTB they probably prevent attack of the mycobacteria by
cationic proteins, lysozyme and oxygen radicals in the phagocytic granule as well as
protecting the extracellular mycobacteria from complement deposition in serum
(Brennan, 1989; Minnikin, 1982).
Cord Factor is responsible for the serpentine cording. The cord factor is toxic
to mammalian cells and is also an inhibitor of polymorphonuclear neutrophils (PMN)
migration. Cord factor is most abundantly produced in virulent strains of MTB
(Jiongwei et al., 1999).
Mycobacterium tuberculosis does not possess the classic bacterial virulence
factors such as toxins, capsules and fimbriae. Tubercle bacillus has special
mechanisms for cell entry; it can bind directly to mannose receptors on macrophages
12
via the cell wall associated mannosylated glycolipid, L-Arabino Muramic acid, or
indirectly via certain complement receptors or Fc receptors (Trias and Benz, 1994).
Intracellular invasion of the host cells is an effective means of evading the
immune system. In particular, antibodies and complement are ineffective once the
microbe is phagocytosed inhibiting phagosome-lysosome fusion. The exact
mechanism used to accomplish this is thought to be the result of a protein secreted by
the bacilli that modifies the phagosome membrane. The bacilli may remain in the
phagosome or escape from the phagosome, in either case finding a protected
environment for growth in the macrophage (Trias and Benz., 1994).
Interferences with the toxic effects of reactive oxygen intermediates produced
in the process of phagocytosis is another method in which compounds including
glycolipids, sulfatides and L-acetyl muramic acid down regulate the oxidative
cytotoxic mechanism and macrophage uptake via complement receptors bypass the
activation of a respiratory burst (Crowle, 1991).
Antigen 85 complex composed of a group of proteins secreted by MTB bind
fibronectin and may aid in walling off the bacteria from the immune system
facilitating tubercle formation (Atlas, 1995). Other factors include the high lipid
content on cell wall and the cord factor (Kubica et al., 1975) which is known to be
toxic to mammalian cells and to be an inhibitor of PMN migration.
13
2.4.4
Pathogenesis of MTB
The droplet nuclei are generated during talking, singing, coughing and
sneezing and remain air-borne for extended periods of time. When the inhaled bacilli
reach the pulmonary alveoli, infection begins in the infected alveolar macrophages
and mycobacteria replicate exponentially (Hopewell, 1994). The Ghon focus is the
primary site of infection in the lungs. However, the bacilli can be picked up by
dendritic cells and transported to the local (mediastinal) lymph nodes and through the
bloodstream to the more distant tissues and organs where TB disease could
potentially develop (Feja, 2005; Schluger, 1994).
Macrophages, T lymphocytes, B lymphocytes and fibroblasts aggregate to
form a granuloma. The granuloma functions not only to prevent dissemination of the
bacilli, but also provides a local environment for communication of cells of the
immune system. Within the granuloma, T lymphocytes (CD4+) secrete a cytokine
such as interferon gamma, which activates macrophages to destroy the bacteria. T
lymphocytes (CD8+) can also directly kill infected cells (Palomino et al., 2007).
When the bacteria are not eliminated with the granuloma, they remain
dormant resulting in a latent infection. Else, the granulomas of human tuberculosis
result in necrosis in the center of tubercles (McDonough et al, 1993).Tissue
destruction and necrosis are balanced by healing and fibrosis. Affected tissue is
replaced by scarring and cavities filled with necrotic material. This material may
therefore be coughed up and contains live bacilli important in transmission of
infection.
14
2.4.5
Transmission of tuberculosis
Transmission of TB occurs primarily by the aerosol route. Coughing by
people with active TB produces droplet nuclei containing infectious organisms which
can remain suspended in the air for several hours and if inhaled infection occurs.
Only 10% of immuno-competent people infected with MTB develop active
disease in their lifetime (Stead, 1981; CDC, 1994) the other 90% do not become ill
and cannot transmit the organism. However, in some groups such as infants or the
immuno-compromised (e.g. those with AIDS or malnutrition) there is a much higher
chance to develop clinical TB (Beck-Sagué et al., 1992). Predisposing factors for TB
infection include: close contact with high populations of people, poor nutrition,
intravenous drug use, alcoholism and HIV infection while the disease progression
however depends on the strain of MTB prior to exposure, vaccination, infectious dose
and immune status of the host (Dye, 2002; Kohsaka, 1993). Factors such as being
homeless, a drug abuser, living in urban areas, and low age have commonly been
found to increase the risk of transmission (Borgdorff, 1999, Borgdorff, 2001, Diel,
2002, Small, 1994, van Soolingen, 1999).
2.4.6
Diagnosis of tuberculosis
Diagnosis is made by a positive tuberculin skin test involving an immune
reaction to a low quantity of tuberculosis antigens present in an individual. It can be
confirmed by chest X-rays and microscopic examination of sputum. Detection of
acid-fast bacilli (using the Ziehl -Neelsen stain) in sputum or tissue samples is
considered a positive diagnosis, however the disease is confirmed by laboratory
15
culture (American Thoracic Society, 1997). The other method of diagnosis of MTB is
molecular diagnosis by gene amplification involving technique polymerase chain
reaction in which a number of commercial kits are available.
2.4.7
Tuberculosis Treatment
There are five first-line drugs used in treatment of tuberculosis this
includes: isoniazid, rifampicin, ethambutol, pyrazinamide, and streptomycin.
(Martindale, 2004; Centers for Disease Control and Prevention 2003). The course of
drug therapy lasts up 6-8 months.
2.4.8
Drug resistance tuberculosis
Drug resistance is a state when MTB strains are resistant to anti-microbial
agents at the level attainable in the blood and tissue (Mitchson, 1985).
Resistant
strains differ from the sensitive strains in their capacity to grow in presence of higher
concentration of a drug. Wild strains are those that have never been exposed to antituberculosis drugs. Naturally resistant strains are wild strains resistant to a drug
without having been in contact with it. It is species specific and has been used as a
taxonomic marker (Jacob, 1994; Chandrasekaran et al., 1990; TDR/SWG, 2005).
Primary resistance develops in persons initially infected with resistant
organisms (Palomino et al., 2007; Krishnaswamy et al., 1976). It includes resistance
in wild strains which have never come into contact with the drug (natural resistance)
with the resistance occurring as a result of exposure of the strain to the drug but in
another patient (Jacobs, 1994). Secondary resistance (acquired resistance) may
develop during TB therapy due to inadequate treatment regimen, i.e. not taking the
16
prescribed regimen appropriately or using low quality medication (Chandrasekaran et
al., 1990). Acquired resistance develops due to exposure of the strain to antituberculosis drugs and the consequent selecting out of resistant mutant bacilli.
However, some of the drug-resistant strains in previously treated patients may
actually represent primary resistance among patients who remain uncured (Frieden,
1993).
Multi-drug resistant TB is defined as resistance to at least two first line TB
drugs: Rifampicin and Isoniazid (WHO/IUATLD, 1997). Initial resistance is the
resistance in patients who give a history of never having received chemotherapy in
the past. It includes primary resistance and resistance to previous treatment concealed
by the patient or of which the patient was unaware (Chandrasekaran et al., 1990;
TDR/SWG, 2005).
Extensively drug resistant (XDR) TB strains have recently emerged. XDRTB strains TB show resistance to at least rifampicin and isoniazid, which is the
definition of MDR-TB, in addition to any fluoroquinolone, and to at least 1 of the 3
following injectable drugs used in anti-TB treatment: capreomycin, kanamycin and
amikacin.
17
2.5Preservation of Microorganisms
2.5.1
Methods of preservation of microorganisms
Certain factors can affect the viability of preserved microorganisms among
them; the method of preservation, nature of the strain, cell concentration and
formulation composition of the storage medium (Macleod and Calcott, 1976; Mazur,
1970; Karow and Critser, 1997; Fuller et al., 2004).
Methods mainly used in the preservation of microorganisms include freeze
drying and cryopreservation. In freeze drying, microorganisms are dried while in a
frozen state. Moisture (ice crystals) is removed as a gas, similar to evaporation in a
process technically called sublimation and the cells do not collapse. This process in
most cases leaves the cellular structure unaltered through out the drying process
(Moore et al, 1975). Sufficiently lowering the temperature to sub zero enhances
viability in dormant state thereby permitting preservation of the strains for extended
periods of time (Grout and Morris, 1987).
Although freeze- drying method can be used in microorganism preservation,
there are some species of Mycobacterium that may prove difficult to freeze-dry
resulting in relatively poor survival rates and if the preservation period of the strain is
very long, viability could be significantly affected (Howard et al., 1967) . However
the optimum preservation condition by freeze drying have been achieved by
suspending mycobacteria either in Dubos Tween-albumin broth or in Middlebrook
7H9 liquid medium supplemented with ADC enrichment and storing at -70 °C
(Kubica et al., 1975) .
18
Disadvantages of using the freeze drying method include; high capital cost of
equipment, high energy costs and long processing time which is typically 24 hour
drying cycle (Rudge et al., 1995). In cryopreservation, cells are preserved by cooling
to very low temperatures, such as -80°C or -196°C the boiling point of liquid
nitrogen. At these low temperatures, any biological activity, including the
biochemical reactions that would lead to cell death is effectively stopped. However,
when vitrification solutions are not used, the cells being preserved are often damaged
due to freezing during the approach to low temperatures or warming to room
temperature (Bhat et al., 2005).
The phenomena which can cause damage to cells during cryopreservation are
solution effects, dehydration and extracellular and intracellular ice formation. When
tissues are cooled slowly, water migrates out of cells and ice forms in the
extracellular space (Kubica et al., 1975). Too much extracellular ice can cause
mechanical damage due to crushing, and the stresses associated with cellular
dehydration can cause damage directly (Grout and Morris, 1987).
Preservation of cultures of mycobacteria by freezing in skimmed milk was
determined to be an easier and more reliable method of maintaining viability and
stability than lyophilization (Kim and Kubica, 1973).
2.5.2
Effects of freezing on microorganisms
Microorganisms vary tremendously in their abilities to tolerate freezing
(Lewis et al., 1993). Environmental factors not withstanding survival is affected by
the type and age of the microorganism. When microorganisms are subjected to an
19
environmental stress such as freezing some cells may express no detrimental effects,
some are killed while some may undergo sub-lethal or metabolic injury. Detrimental
effects of freezing on microbial cells occur due to: thermal (cold) shock,
concentration of extracellular solutes, toxicity of concentrated intracellular solutes,
cell dehydration, internal ice formation and attainment of minimum cell volume
(Calcott and MacLeod, 1974).
Two kinds of lethal effects to microorganisms that occur during freezing are
the immediate direct consequence of freezing and thawing as well as killing during
frozen preservation (Schmidt-Lorenz, 1976). Cold shock responses are common when
microbes are in the exponential phase of growth and are suddenly exposed to low
temperatures (Ingram and Buttke, 1984).
During the freezing process, aqueous solutions remain in their liquid phase
until reaching their freezing point at temperatures below 0˚C (Corry, 1987). When the
suspensions are cooled to temperatures below 0˚C, both the suspending medium and
the cells are initially supercooled (Mazur, 1970). The cells suspended in aqueous
solutions behave like solutes molecules. They become concentrated in the unfrozen
portion of the solution as ice crystals form (Brown, 1991). In the freezing process,
extra-cellular ice crystal formation precedes intracellular freezing point of the
suspending medium and presence of ice nucleating agents (Steponkus, 1984). This
intracellular supercooling can be explained by the fact that the cell membrane
prevents growth of the extracellular ice into the cell interior, and the cell itself
apparently does not contain nucleators of supercooled water (Mazur, 1965).
20
As long as supercooling of the cell suspending medium lasts and extracellular
ice crystal formation has not initiated, cells are subject to a drop in temperature.
Damage to the cellular structures and inhibition of cellular functions occurs in these
conditions (Grout et al., 1990). Cell viability seems hardly affected indicating that the
temperature drop is not particularly detrimental to the cells (Lorenz, 1974; Douzou,
1982; Park et al., 1997).
Upon extracellular freezing, cells are entrapped between ice crystals and are
subjected to mechanical and adhesion stress (Grout et al., 1990). Formation of the
intracellular ice crystals on the other hand can cause distortion of the membrane
integrity because they are small enough to distort the membrane (Fraizer and
Westhoff, 1978; Brown, 1991). The damage of the cell membrane results in leakage
of the intracellular constituents and loss of the ability to maintain the internal
environment.
Both water outflow and intracellular ice crystal formation are associated with
cellular injury to the plasma membrane and the cell wall since cells are not limitlessly
elastic to be able to shrink to a certain volume without injury (Wolfe et al., 1985).
Upon cell shrinkage plasma membrane material is released via endocytolic
vesculation or exototic extrusion, and the cell wall density increases (Steponkus,
1984; Morris et al., 1998).
Membrane damage is more detrimental than cell wall damage (Calcott and
MacLeod, 1975), the intracellular ice crystals are believed to rupture the plasma
membrane, resulting in release of cellular components into the environment (Mazur,
21
1965, 1977) and the surface membrane is often considered the primary site of
freezing injury (Souzu, 1989). In addition the generation of electrical fields and gas
bubbles in association with the ice fronts have been reported, which is possibly
injurious to the cell due to mechanical damaging of membranes (Kruuv et al., 1985;
Grout et al., 1990; Morris et al., 1988)
The freezing rate is proportional to the size of the ice crystals formed. That is
the faster a solution is frozen the lower the size of the crystal. Slow freezing supports
the formation of large extracellular ice crystals, while small intracellular crystals
develop during faster rates of freezing (Jay, 1996). The mechanisms by which
freezing causes cell damage include efflux of water from the cell, resulting in
precipitation of cytoplasmic solutes and components (Mazur, 1970), mechanical
stress to cellular components, and rupture of cell membranes due to ice crystal
formation (Mazur, 1977; Souzu, 1989).
2.5.3
Effects of thawing on microorganisms
As ice crystals grow they exert the same physical stress on microbial cells
during the freezing process. In addition as the frozen solution begins to melt the
medium surrounding the microbial cells is diluted and cells are exposed to osmotic
shock and the rate of thawing has little effect on survival of microorganisms that have
been previously frozen (Calcott and Thomas, 1978; Lewis et al., 1993)
22
2.6Medium used in preservation
2.6.1
Gelatin
Gelatin is a protein that does not occur in nature but produced by partial
hydrolysis of collagen extracted from connective tissues of animals. It is formed
when natural molecular bonds between individual collagen strands are broken down
into a form that rearranges more easily (Veis, 1964). There are two types of gelatin
dependent on whether or not the preparation involves an alkaline pretreatment, which
converts asparagine and glutamine residues into their respective acids and results in
higher viscosity. Acid pretreatment (Type A gelatin) uses pig skin whereas alkaline
treatment (Type B gelatin) makes use of cattle hides and bones (Schrieber, 2007).
Gelatin is amorphous and therefore lacks a defined structure; it is a
heterogeneous mixture of single or multi-stranded polypeptides, each with extended
left-handed proline helix conformations containing between 300 - 4000 amino acids.
The triple helix of type I collagen extracted from skin and bones is composed of two
α1 (I) and one α2 (I) chains, each with molecular mass ~95 kD, width ~1.5 nm and
length ~0.3 μm. The heterogeneous mixture contains many proline and 4hydroxyproline residues. A typical structure is -Ala-Gly-Pro-Arg-Gly-Glu-4Hyp-GlyPro-.
Gelatin can be dispersed in a relatively concentrated acid. Such dispersions
are stable with little or no chemical changes for 10-15 days. Gelatin is also soluble in
most polar solvents. The mechanical properties are very sensitive to temperature
variations, as well as the medium concentration which have important effects on
23
viscosity. The higher they are, the higher the viscosity obtained (Ward and Courts,
1977).
Figure 2:
2.6.2
Molecular structure of gelatin
Monosodium glutamate (MSG)
Monosodium glutamate (MSG) is the sodium salt of glutamate produced
through fermentation processes using molasses from sugar cane or sugar beet, as well
as starch hydrolysates from corn, or tapioca (Giacometti, 1979). Prior to the
development of the fermentation process, it was produced by hydrolysis of natural
proteins including wheat gluten and defatted soybean flakes. It is a white crystalline
powder readily soluble in water but sparingly soluble in ethanol with a molecular
weight of 187.13 and a melting temperature of 225°C. By being stable it does not
change in appearance or quality during prolonged preservation at room temperature.
MSG is partially dehydrated and converted into 5-pyrrolidone-2-carboxylate at acidic
conditions (pH 2.2-2.4) and at high temperatures (Lehninger, 1982; Meister, 1979).
Monosodium glutamate (MSG) has been reported to be an efficient protectant,
an increase in residual activity and viability during freeze drying following the
24
addition of MSG to the drying medium has been previously reported for various
organisms (Carvalho et al, 2003; Yoo et al., 1993).
Figure: 3
2.6.3
Chemical structure of MSG
Trypticase Soy Broth with 20% Glycerol
Trypticase Soy Broth (Soybean-Casein Digest Medium) is a nutritious
medium that supports the growth of a wide variety of microorganisms, including
common aerobic, facultative and anaerobic bacteria and fungi (MacFaddin, 1985). It
is a general-purpose medium used in qualitative procedures for the cultivation of
fastidious and non fastidious microorganisms from a variety of clinical and non
clinical specimens.
Trypticase Soy Broth is used in the long-term frozen maintenance of bacterial
stock cultures it is supplemented with glycerol, may be used as a maintenance
medium for the preservation of bacterial cultures (Kirsop and Snell, 1984).
25
2.6.4
Glycerol
Glycerol is a trihydric alcohol with three hydrophilic alcoholic hydroxyl
groups that are responsible for its solubility in water and its hygroscopic nature. This
sugar alcohol has a fittingly sweet-taste and of low toxicity. It is a colorless, odorless
and viscous liquid with a surface tension of 64.00 at 20 °C in mN/m with a
temperature coefficient of -0.0598 mN/ (m K). It is a central component of lipids and
used as antifreeze or a cryoprotectant in cryogenic processes. It can diffuse through
the lipid membranes both in vitro (Paula et al., 1996) and in vivo (Eze and
McElhaney, 1981),
2.6.5
Sterilized distilled water
Distilled water is water that has virtually all of its impurities removed through
distillation it involves a process of boiling the water and re-condensing the steam
leaving most if not all solid contaminants behind. It allows sufficiently pure water for
some applications such as the attempt to create sterile, enzyme-free medium.
The pH of distilled water is 7.0 however distilled water may have a pH that is slightly
acidic (less than 7.0) due to the presence of carbon dioxide (CO2) that is absorbed
from the atmosphere. Dissolved carbon dioxide reacts slowly with water to give the
bicarbonate and hydronium ions.
CO2 + 2H2O
HCO3- + H3O+
During distillation, the dissolved CO2 is driven out of the liquid while during
condensation the water re-absorbs the CO2 again resulting in a pH that is less than
7.0.
26
2.6.6
Phosphate buffer saline (PBS)
Phosphate buffer is used in the preparation of dilution blanks for use in
microbiological testing rather than unbuffered water in order to standardize potential
variable due to the wide variation in the pH of purified water from multiple sources.
PBS has many uses because it is isotonic and non-toxic to cells and can be used to
dilute substances as well as a cellular cleaning solution which ensures prolonged drypreservation of immobilized-biomolecules like proteins and enzymatic proteins.
PBS is used as biomolecule diluent since it can structure water around
biomolecules immobilized to the solid surface. Such thin film of water prevents
denaturing of biomolecules or conformational changes to them. Carbonate buffers
may be used for the same purpose but with less effectiveness.
2.6.7
Lactose
Milk sugar or lactose is a disaccharide (C12H22O11) that is found only in milk.
This carbohydrate exists in two isomeric forms. Both forms can crystallize but the
physico-chemical relationships between the different forms of lactose are very
complex
The disaccharide consists of β-D-galactose and β-D-glucose molecules bonded
through a β1-4 glycosidic linkage. Lactose makes up around 2-8% of the solids in
milk (Jenness and Koops, 1962)
It has a melting point of a range of 200-202 °C and Solubility of 1 in 4.63
measured %w/v. This translates to 0.216g of lactose dissolving readily in 1mL of
water. The solubility of lactose in water is 18.9049 g at 25 °C, 25.1484 g at 40 °C and
27
37.2149 g at 60 °C per 100 g solution. Its solubility in ethanol is 0.0111 g at 40 °C
and 0.0270 g at 60 °C per 100 g solution (Nickerson et al,1974)
Figure 4:
Molecular structure of lactose
28
CHAPTER THREE
3.12
METHODOLOGY
3.13
Study design
The study design was a prospective laboratory based study in which the recovery rate of
Mycobacterium tuberculosis strains preserved at different temperatures and media was
determined.
3.14
Study site
The study was laboratory based and was carried out at the Center for Respiratory Disease
Research (CRDR- KEMRI) laboratories
3.15
Study population, inclusion and exclusion criteria
In this study only MTB strains were used and were acquired from the Center for Respiratory
Diseases Research archives.
Mycobacteria other than tuberculosis (MOTTS) were excluded from the study.
3.16
Sampling
The sample size was determined by modification of Habeenzu et al., (1999) formulae: a
recovery rate of 93% was used with a 95% confidence interval and 5% error margin.
n = ((Z/2) 2 pq) / d2
Where: (Z/2) 2 is the corresponding value to the 95% confidence interval
n= sample size
p = the recovery rate
q = 1- p
d is the allowable error margin
Therefore:
n = (1.96) 2 (0.93) (0.7) = 100.0353  100 samples
(0.05) 2
29
3.17
Preparation of Preservation media
3.17.1 Trypticase Soy Broth + 20% Glycerol
The Trypticase Soy Broth Dextrose 27.5g medium was suspended in 800ml
distilled or deionized water in Erlenmeyer flasks in which 200ml glycerol was topped
to the 1000ml mark. The mixture was gently heated with agitation to dissolve the
constituents to make a final pH, 7.3 ± 0.2. The medium was dispensed in 20ml
universal glass bottles and autoclaved at 121°C for 15 min then cooled to room
temperature and stored at 4˚C.
3.17.2 OADC enriched Middlebrook 7H9 Broth (BD) + 5% glycerol
4.7g of Middlebrook 7H9 Broth powder was suspended in 850 ml of purified
water in which 50 ml glycerol was added. The mixture was autoclaved at 121˚C for
15 min. Aseptically 100 ml of Middlebrook OADC Enrichment added to the medium
when it cooled to room temperature and 1ml dispensed directly 2ml cryovials.
3.17.3 Middlebrook 7H9 Broth (BD)
4.7g of Middlebrook 7H9 Broth powder was suspended in 1000 ml of
sterilized distilled water. The mixture was autoclaved at 121˚C for 15 min. The
medium was dispensed in 20ml universal glass bottles.
3.17.4 Lactose broth Difco™
150g of lactose powder was dissolved in 1000ml of sterilized distilled water
then dispensed in universal glass bottles, in 20 ml amounts and autoclaved at 121°C
for 10 min at 10 pounds pressure or tyndallized to reduce the hydrolysis of lactose.
After autoclaving, the broth was cooled quickly.
30
3.17.5 Phosphate Buffered Saline BBL™
Monobasic sodium phosphate was dissolved into 280ml sterilized distilled
water into which 720ml of dissolved dibasic sodium phosphate was added, 9grams
Saline (NaCl) was added to make a pH is 7.4.
3.17.6 Skim Milk Medium BBL™
100g skim Milk powder was dissolved in 1000 distilled water and warmed
to completely dissolve the powder. The medium was dispensed in 20ml universal
glass bottles and autoclaved at 121˚C for 10 min.
3.17.7 Sodium glutamate medium
5g Sodium glutamate was weighed into a sterile flask, and then dissolved with
100ml sterilized distilled containing 6ml glycerol by heating. The solution was
dispensed in 20ml universal glass bottles and autoclaved at 121˚C for 30 min after
which it was cooled to room temperature before preservation in a refrigerator.
3.17.8 Lowenstein Jensen medium (LJG)
3.17.8.1
Mineral salt solution
4 g Potassium dihydrogen phosphate anhydrous (KH2PO4), 0.4g Magnesium
sulphate (MgSO4.7H2O), 1g Magnesium citrate, 6g L-Asparagine, 20ml Glycerol
(reagent grade) and 1000ml distilled water were measured to constitute the mineral
salts solution.
31
Into a sterile 2000ml volumetric flask the reagents were dissolved completely
by heating with occasional swirling. The resulting solution was autoclaved at 1210C
for 30 min to sterilize then stored in a refrigerator at (40C) after cooling to room
temperature.
3.17.8.2
Malachite green solution (2%)
2.0g Malachite green dye was dissolved completely in 100ml distilled water
and aliquoted in 20mls volume into universal bottles before autoclaving.
3.17.8.3
Egg base
Fresh eggs (up to 3 days old) were soaked for 5 min in plain devo clean
solution, cleaned by gently scrubbing with a hand brush and thoroughly rinsed in
running tap water and then allowed to dry.
The egg shells were wiped with cotton wool soaked in methylated spirit and
cracked on the edge of the graduated glass jar and poured into a sterile 6 Liter
volumetric flask with sterile 3mm glass beads to make 500mls. The jar was shaken
vigorously to break eggs fully.
3.17.8.4
Lowenstein Jensen media preparation
Aseptically 20ml malachite green solution and 600ml mineral salt solution
were added into the 6000ml volumetric flask containing eggs then shaken well
before sieving the mixture and adding penicillin drug in the ratio of 1ml: 1000ml of
egg based media. 5 ml of the solution was dispensed into universal bottles and
inspissated at 850C for 1 hour. The finished product was tested of for performance
(see appendix).
32
3.18
Subculture of stored strains
MTB Strains were selected from the CRDR archives from which a loopful of
MTB cells were inoculated on freshly prepared Lowenstein Jensen media and
incubated at 37˚C for four weeks. After confluent growth was established on the slope
the cells were aliquoted into cryovials containing the different preservation medium
and preserved at respective preservation temperatures.
3.19
Preservation suspension
MTB strains were harvested by scraping cells on the growth surface of the
Lowenstein Jensen media from which the cellular suspensions were prepared.
Approximately 4 mg moist weight of a representative sample of the bacterial mass
visualized as 2/3 loopful of 3mm internal diameter 24 SWG wireloop was added into
1 ml of sterile liquid preservation medium in a 2 ml cryovial. Two sets of each strain
were preserved. One was to be utilized in approach one while the other in approach
two.
3.20
Freezing and thawing of MTB strains
3.20.1 Approach one
3.20.1.1
Direct freezing
Strains were preserved using a single step (direct) method to the respective
temperature. That is to RT, or 4˚C, or -20˚C, or -80˚C.
3.20.1.2
Direct thawing
Strains preserved at -80ºC, -20˚C and 4˚C were thawed directly to room
temperature for one hour, vortexed for 1min before inoculation on Lowenstein Jensen
33
and incubation at 37°C. Growth on the slopes was graded and recorded after four
weeks incubation.
3.20.2 Approach two
3.20.2.1
Systematic freezing
Strains were preserved in stepwise method with an interval of one hour on
each step starting from RT to 4˚C, to -20˚C, and finally to -80˚C.
3.20.2.2
Systematic thawing
The systematic method of thawing started from the lowest preservation
temperature through to RT (e.g. -80˚C to -20˚C, to 4˚C finally to RT with an interval
of one hour on each step). Strains were subcultured on LJG media and incubated at 37
°C. Growth on the slopes was graded and recorded after four weeks incubation.
3.21
Inoculation of preserved suspension
Each preserved isolate in the cryovial was vortexed to produce a uniform
suspension from which one loopful (3mm diameter, 27 SWG) was inoculated on the
LJ medium and incubated for four weeks prior to grading of growth on the slopes.
Subcultures for the five consecutive subsequent months were done by the repeat of
the latter procedure
3.22
Reading of cultures
The strains subcultured on freshly prepared LJ slopes were graded after the
fourth week of incubation at 37ºC (see appendix).
34
3.23
Statistical analysis
Cross-tabulation of grading of growth was performed to determine
interrelation between temperature and media using Middlebrook 7H9 as the standard.
Correlation coefficient of concordance of grading was performed to quantify the
effect of the nine suspension media on viability of MTB strains in four preservation
temperatures during five months of preservation.
Significant differences within and between the grading of each suspension
media were determined by using the Kendall’s coefficient of concordance. SPSS
Science, Chicago, IL (version 11.5) software was used.
35
CHAPTER FOUR
4.0 RESULTS
After random selection, 97 MTB strains were subcultured and incubated at
37ºC four weeks. Growth on the surface of the LJ slope was graded (see appendix)
for the 97 strains. Where by 91 (93.8%) strains had a grading of 3+, 5 (5.2%) strains
had a grading 2+ while 1 (1%) had a grading 1+.
From these subcultures, the strains were aliquoted into the nine different
preservation suspensions then subcultures were made from the suspensions before
preservation. Results from these subcultures (initial grading) were correlated with
growth grading of subsequent months to determine intra-media variability as shown
on figures 5 to 13.
Ranking of the different suspension media was done using the standard medium
(7H9) initial grading with the grading after five months of preservation as shown on
figures 14 to 17.
In determining the effect of the media and temperature after direct freezing and
thawing, results obtained from the approach one initial subculture grading were
correlated with grading after the fifth month of preservation as shown on figure 18.
To determine if there was any significance difference between the two approaches
used in preservation, grading from direct approach was correlated with grading from
systematic approach month five and results are shown on figure 19.
36
4.1 Monthly MTB growth recovery for 5% sodium glutamate (SG)
Figure 5 shows the percentage of viable organisms concordant with the initial
subculture reading between SG initial and SG for five subsequent months of
subculture at -80, -20ºC, 4ºC and RT.
There was a large concordance (τ>0.6) at the temperatures -80ºC, -20ºC, and
4ºC. Room temperature had large concordance up to the third month but medium
concordance for month five.
There was no significance difference (p<0.05) in growth at all temperatures
during the preservation period.
100%
90%
y = -0.0742Ln(x) + 0.9577
R2 = 0.2162
Growth (% viable)
80%
70%
60%
50%
40%
30%
20%
10%
(1
T)
T)
(R
5
T)
(R
4
T)
(R
3
T)
(R
(R
2
1
(4
ºC
)
(4
ºC
)
3
(4
ºC
)
4
(4
ºC
)
5
(4
ºC
)
2
1
2
80
º
(- C)
80
3
( - ºC
8
4 0 ºC
(8
5 0º
(C
80
ºC
1
(2
2 0 ºC
(2 )
3 0 ºC
(2 )
4 0 ºC
(2 )
5 0 ºC
(20 )
ºC
)
0%
Month
Figure 5: Monthly MTB growth recovery on 5% sodium glutamate (SG)
37
4.2 Monthly MTB growth recovery on skimmed milk (SM)
Figure 6 shows the percentage of viable organisms concordant with the initial
subculture reading between initial SM and five subsequent months grading at -80, 20ºC, 4ºC and RT.
Preservation at -80ºC had high concordance (τ>0.7) for all the five months of
preservation. Whereas -20ºC had high concordance (τ>0.6) in the first, second, third
and fourth months but low concordance (τ=0.2) on the fifth month.
At 4ºC there was high concordance value (τ>0.6) on all the five months of
preservation. At room temperature there was high concordance on the first, second,
third and fourth months but low concordance on the fifth month of preservation.
Even with varying concordance, there was no significant difference (p<0.05)
for the five months of preservation in the temperatures used.
38
90%
80%
70%
60%
50%
40%
30%
20%
y = -0.1114Ln(x) + 0.9429
R2 = 0.2951
(R
2 T)
(R
3 T)
(R
4 T)
(R
5 T)
(R
T)
10%
1
0%
Month
(8
2 0 ºC
()
3 80
(- ºC
4 80
(- ºC
5 80
(- ºC
80
ºC
1
(2 20
(- ºC
3 20 )
(- ºC
4 20 )
(- ºC
5 20 )
(- ºC
20 )
ºC
)
1
(4
2 ºC)
(4
3 ºC)
(4
º
4 C)
(4
5 ºC)
(4
ºC
)
100%
Growth (%viable)
1
Figure 6: Monthly MTB growth recovery on skimmed milk (SM)
39
4.3 Monthly MTB growth recovery on 7H9 OADC-enriched Middlebrook 7H9
medium + 5%glycerol (7H9 OADC)
Figure 7 shows the percentage of viable organisms concordant with the initial
subculture reading between initial 7H9 OADC and five consecutive months grading
at -80, -20ºC, 4ºC and RT. The preservation temperature of -80ºC had a high
concordance (τ>0.8) for the five consecutive months of preservation, while -20ºC had
high concordance value (τ>0.8) for the first, second, third and fourth months but
medium concordance for the fifth month.
Strains preserved at 4ºC had high grading concordance (τ>0.8) for the first
second, third and fourth months but low concordance on the fifth month. At RT, there
was high concordance value (τ>0.6) in the first, second, and third months but low
concordance in the fourth and fifth months.
There was no significant difference (p<0.05) in grading for the five months of
preservation in the temperatures used.
40
Growth (% viable)
100%
90%
80%
70%
60%
50%
40%
30%
20%
Month
) C C C C
) ) ) ) )
ºC º º º º
ºC ºC ºC ºC ºC
0
0
0
0
80 - 8 - 8 - 8 - 8
20 20 20 20 20
(- 2 ( 3 ( 4 ( 5 (
(- (- (- (- (1 2 3 4 5
0%
10%
1
1
1
T) T) T) T) T)
(R (R (R (R (R
2 3 4 5
y = -0.1415Ln(x) + 1.1119
R2 = 0.361
) ) ) ) )
ºC ºC ºC ºC ºC
(4 (4 (4 (4 (4
2 3 4 5
4.4 Figure7: Monthly MTB growth recovery on 7H9 OADC
41
4.5 Monthly MTB growth recovery on Middlebrook 7H9 (7H9)
Figure 8 shows the percentage of viable organisms concordant with the initial
subculture reading between initial 7H9 and grading for five subsequent months at -80,
-20ºC, 4ºC and RT.
Preservation at -80ºC showed high concordance in all the five months of
preservation (τ> 0.6) while preservation at -20ºC showed high concordance (τ>0.6)
on the first, second and fourth months. The third and fifth months showed low
concordance.
Preservation at 4ºC showed medium concordance value in the first, second
and third months, while the fourth and fifth month showed low concordance. Room
temperature showed a high concordance for the first month and low concordance
(τ=0.4) in the second third, fourth and fifth months. There was no significant
difference (p<0.05) in grading in the five months of preservation in the temperatures
used.
42
Growth (% viable)
100%
90%
80%
70%
60%
50%
40%
30%
20%
Month
2 80
( - ºC
3( 80 )
- ºC
4 80 )
(- ºC
5 80 )
( - ºC
80 )
ºC
)
1
(2 20
( - ºC
3 20 )
(- ºC
4 20 )
( - ºC
5 20 )
(- ºC
20 )
ºC
)
1
(4
2 ºC)
(4
3 ºC)
(4
4 ºC)
(4
5 ºC)
(4
ºC
)
1
(R
2 T)
(R
3 T)
(R
4 T)
(R
5 T)
(R
T)
0%
(-
10%
1
y = -0.171Ln(x) + 0.8548
R2 = 0.4849
Figure 8: Monthly MTB growth recovery on Middlebrook 7H9
43
4.6 Monthly MTB growth recovery on 15% aqueous solution of lactose at pH 5.0
(lactose)
Figure 9 shows the percentage of viable organisms concordant with the initial
subculture reading between initial lactose and grading for five subsequent months at 80, -20ºC, 4ºC and RT.
The preservation temperature -80ºC had high concordance (τ>0.7) in the first,
second, and third months. The fourth and fifth month showed low concordance.
Preservation at -20ºC showed high concordance (τ>0.6) for the first four months and
low concordance on the fifth month. 4ºC had high concordance value (τ>0.6) for the
first, second, and third months. The fourth showed medium concordance while the
fifth month had low concordance. Room temperatures showed high concordance
(τ>0.7) in the first month but low concordance in the second, third, fourth and fifth
months.
There was no significant difference (p<0.05) in grading in the five months of
preservation in the temperatures used.
44
Growth (% viable)
100%
90%
80%
70%
60%
50%
40%
30%
20%
(-
)
)
)
)
)
)
)
)
)
)
ºC ºC ºC ºC ºC
ºC ºC ºC ºC ºC
80 - 80 - 80 - 80 - 80
20 - 20 - 20 - 20 - 20
(
(
(
(
((
(
(
(
2
3
4
5
1
2
3
4
5
0%
10%
1
Month
1
)
)
)
)
)
ºC ºC ºC ºC ºC
(4 (4 (4 (4 (4
2
3
4
5
(R
T) T) T) T) T)
(R (R (R (R
2
3
4
5
y = -0.1764Ln(x) + 0.9506
R2 = 0.3746
1
Figure 9: Monthly MTB growth recovery on lactose
45
4.7 Monthly MTB growth recovery on 1% gelatin buffered at pH 6.8 (gelatin)
Figure 10 shows the percentage of viable organisms concordant with the
initial subculture reading between initial gelatin and grading for five subsequent
months at -80, -20ºC, 4ºC and RT.
The preservation temperature -80 ºC had high concordance (τ>0.6) in all
months. The -20ºC temperature had high concordance in the first four months (τ >0.6)
and low concordance (τ=3) on the fifth month. 4ºC had high concordance (τ=0.8) on
the first month. The second, third and forth months had medium concordance while
the fifth month had low concordance value (τ<0.2). Room temperature showed high
concordance on first and second months, while third, forth and fifth months had low
concordance. There was no significant difference (p<0.05) in grading in the five
months of preservation in the temperatures used.
46
Growth (% viable)
100%
90%
80%
70%
60%
50%
40%
30%
20%
(-
1
1
T) T) T) T) T)
(R (R (R (R (R
2 3 4 5
y = -0.1994Ln(x) + 1.0244
R2 = 0.4783
) ) ) ) )
ºC ºC ºC ºC ºC
(4 (4 (4 (4 (4
2 3 4 5
Month
) ) ) ) )
) ) ) ) )
ºC ºC ºC ºC ºC
ºC ºC ºC ºC ºC
80 - 80 - 80 - 80 - 80
20 20 20 20 20
( ( ( (
(- (- (- (- (2 3 4 5
1 2 3 4 5
0%
10%
1
Figure 10: Monthly MTB growth recovery on gelatin
47
4.8 Monthly MTB growth recovery on Phosphate buffered saline pH 7.2
Figure 11 shows the percentage of viable organisms concordant with the
initial subculture reading between PBS initial verses and grading for five subsequent
months at -80, -20ºC, 4ºC and RT.
Strains preserved at -80ºC showed high concordance (τ>0.6) in the five
subsequent months of preservation. Strains preserved at -20ºC had high concordance
(τ>0.6) in the first, second, and third months medium concordance on the fourth
month while low concordance (τ=0.3) in fifth month.
Preservation at 4ºC showed only high concordance (τ=0.8) in the first month
but with low concordance (0.2<τ<0.5) third, fourth and fifth months.
There was no significant difference (p<0.05) in grading in the five months of
preservation at -80, -20ºC and 4ºC.
Preservation at RT only showed a high concordance on the first month (τ>0.7)
while the second, third and forth months had low concordant values. Grading on the
fifth month showed low discordance and had a significant difference (p<0.05).
48
Growth (% viable)
110%
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
y = -0.2118Ln(x) + 0.9929
R2 = 0.4807
-10% ) ) ) ) )
) ) ) ) )
) ) ) ) )
) ) ) ) )
ºC ºC ºC ºC ºC
ºC ºC ºC ºC ºC
ºC ºC ºC ºC ºC (RT (RT (RT(RT (RT
1 2 3 4 5
80 80 80 80 80 -20 -20 -20 -20 -20 1(4 2(4 2(4 4(4 5(4
(- (- (- (- (( ( ( ( (
1 2 3 4 5
1 2 3 4 5
Month
Figure 11: Monthly MTB growth recovery on PBS
49
4.9 Monthly MTB growth recovery on trypticase soy broth + glycerol (TSB)
Figure 12 shows the percentage of viable organisms concordant with the
initial subculture reading between TSB initial and grading for five subsequent months
at -80, -20ºC, 4ºC and RT. Strains preserved at -80ºC had high concordance (τ>0.8).
When preserved at -20ºC there was high concordance (τ>0.6) on the first, second,
third and fourth months. Medium concordance value for the fifth month (τ=0.5).
There was no significant difference (p<0.05) in all the five months of preservation for
temperatures -80ºC, -20ºC, and 4ºC.
At room temperature there was a significant difference (p<0.05) in grading in
the fourth and fifth months of preservation (τ<-0.3) and low concordance grading in
the first, second and third months (-0.4<τ<0.5).
50
Growth (% viable)
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
y = -0.3503Ln(x) + 1.3564
R2 = 0.5368
1(
R
2( T)
R
T
3( )
R
4( T)
R
5( T)
R
T)
0%
-10%
-20%
-30%
-40%
Month
1(
-8
2( 0ºC
-8 )
0
3( ºC
80 )
4( ºC
-8 )
5( 0ºC
-8 )
0º
C
)
1
(2 20º
(- C
3 20º )
(- C
2 )
4 0º
(- C)
5 20º
(- C
20 )
ºC
)
1(
4º
2( C)
4º
3( C)
4º
4( C)
4º
5( C)
4º
C
)
Figure 12: Monthly MTB growth recovery on TSB
51
4.10
Monthly MTB growth recovery on sterilized distilled water (SDW)
Figure 13 shows the percentage of viable organisms concordant with the
initial subculture reading between initial SDW and grading for five subsequent
months at -80, -20ºC, 4ºC and RT.
Preservation at -80ºC and -20ºC showed high concordance in first three
months of preservation (τ> 0.6) but low concordance on the fourth and fifth months.
Preservation at 4ºC and RT showed high concordance on the first and second but low
concordance on the third, fourth and fifth months. There was no significant difference
(p<0.05) in grading in the five months of preservation in the temperatures used.
100%
Growth (% viable)
90%
80%
70%
y = -0.1787Ln(x) + 0.9177
R2 = 0.2761
60%
50%
40%
30%
20%
10%
1(
RT
2( )
RT
3( )
RT
4( )
RT
5( )
RT
)
1(
-8
2( 0ºC
-8 )
0
3( ºC)
80
4( ºC
-8 )
5( 0ºC
-8 )
0º
C)
1
(-2
2 0ºC
(-2 )
3 0ºC
(-2 )
4 0ºC
(-2 )
5 0ºC
(-2 )
0º
C)
1(
4º
2( C)
4º
2( C)
4º
4( C)
4º
5( C)
4º
C)
0%
Month
Figure 13: Monthly growth recovery for MTB on SDW
52
4.10
Growth grading concordance for media at -80ºC
Figure 14 shows the correlation between grading of initial Middlebrook 7H9
(control medium) and grading for all preservation media on the fifth at -80ºC.
According to the following order; Sodium glutamate, trypticase soy broth, 7H9
Middlebrook, and OADC enriched Middlebrook 7H9 had high concordance (τ>0.5).
Skimmed milk, gelatin, phosphate buffered saline lactose, and sterilized distilled
water media had a concordance of (0.1<τ<0.5).
There was no significant difference (p<0.05) in grading between the control
and the tested media.
Figure 14: Growth grading concordance for media at -80ºC
53
4.11 Growth grading concordance for media at -20ºC
Figure 15 shows the correlation between initial Middlebrook 7H9 and grading
for all preservation media on the fifth at -20ºC.
There was low concordance (τ<0.5) in the correlation with all media. The
following order shows how the media were ranked according to their concordance;
Sodium glutamate, trypticase soy broth media, OADC enriched Middlebrook 7H9,
7H9 Middlebrook, lactose and gelatin. There was no significant difference (p<0.05)
in grading between the control and these media.
However, there was significant difference (p<0.05) for skimmed milk,
phosphate buffered saline and sterilized distilled water.
Figure 15: Growth grading concordance for media at -20ºC
54
4.12
Growth grading concordance for media at 4ºC
Figure 16 shows cross tabulation of grading for initial Middlebrook 7H9 and
grading for all media month five at 4ºC in which there was no significant difference
(p<0.05) in grading for Sodium glutamate high concordance, skimmed milk and
OADC enriched Middlebrook 7H9.
Trypticase soy broth media, 7H9 Middlebrook, sterilized distilled water,
gelatin, lactose, and phosphate buffered saline media which had low concordance
(τ>0.2) and a significant difference (p<0.05).
Figure 16: Growth grading concordance for media at 4ºC
55
4.13
Growth grading concordance for media at room temperature
Figure 17 shows correlation of grading between initial Middlebrook 7H9 and
grading for all media at month five. At this temperature there was low concordance in
all media apart from PBS and TSB which showed small discordance. No significant
difference (p<0.05) was observed for skimmed milk, Sodium glutamate, OADC
enriched Middlebrook 7H9 lactose and gelatin. However significant difference
(p<0.05) was observed in sterilized distilled water, 7H9 Middlebrook, phosphate
buffered saline, and trypticase soy broth with concordance (τ>0.1).
Figure 17 Growth grading concordance for media at room temperature
56
4.14
Effects of direct freezing and direct thawing in recovery of strains
Figure 18 shows growth recovery results for direct freezing and direct
thawing. At -80 ºC OADC-enriched Middlebrook 7H9 + 5%glycerol had the highest
yield while sterilized distilled water had the lowest yield (p<0.05). At -20ºC 5%
sodium glutamate + glycerol had highest yield while sterilized distilled water had the
lowest yield p<0.05. At 4ºC 5% sodium glutamate + glycerol had highest yield while
phosphate buffered saline pH 7.2 had the lowest yield (p<0.05). At RT skimmed milk
had the highest yield while trypticase soy broth + glycerol had the lowest grading
p<0.05.
57
7H
9
Growth
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Media and temperature
O
AD
C
TS - 80
B ºC
-8
G SG 0ºC
el
at - 80
in ºC
SM 80 º
PB -8 C
La S - 0ºC
ct 80
os C º
C
SD e -8
0
W ºC
-8
0º
C
7H
9 SG
O
AD - 20
C C ºC
T 20
G SB C ºC
el
at -20
in ºC
-2
La SM 0 ºC
ct
os -2 0º
C
e
PB -20
S ºC
SD - 2
W 0ºC
-2
0º
C
7H
9 SG
O
A 4º
G DC C
el
a 4
La t in ºC
ct 4º
os C
e
TS 4ºC
B
4
SM ºC
PB 4ºC
S
SD 4 º
W C
4º
C
Figure 18: Direct freezing and direct thawing in recovery of strains
58
4.14
Comparison of recovery between approach one and approach two
Figure 19 shows the correlation between the grading of strains recovered from
-80ºC by approach one (dt) and approach two (st).
There was a high concordance (τ>0.6), with no significant difference (p<
0.05) in all media in either of the approach used. Sterilized distilled water had the
lowest concordance in grading while OADC-enriched Middlebrook 7H9 medium +
5%glycerol, skimmed milk, 1% gelatin buffered at pH 6.8, trypticase soy broth +
glycerol, 5% sodium glutamate, Middlebrook 7H9, Phosphate buffered saline pH 7.2,
and 15% aqueous solution of lactose at pH 5.0 had higher grading in descending
order.
1.0
0.9
Concordance in grading
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
dt
dt
W
SD
s
S
D
W
s
st
V
La
ct
os
B
S
P
st
V
La
ct
os
e
P
B
S
st
Vs
st
Vs
7H
9
st
e
dt
dt
7H
9
dt
G
Vs
S
e
G
G
el
at
in
e
S
G
el
at
in
s
st
V
B
TS
dt
dt
B
TS
st
V
st
V
S
M
9
7H
Vs
st
O
A
D
C
7H
9
s
s
S
O
A
D
M
C
dt
dt
0.0
Media
Figure 19: Correlation of recovery between approach one and approach two in
recovery of MTB
59
CHAPTER FIVE
5.0
DISCUSSION
The major problem for microbiologists is the maintenance and preservation of
bacterial cultures. Convenient methods for maintaining the viability of cultures in a
stable genetic form and general characteristics are necessary. The choice of
preservation methods is related to the nature of the microorganism, period of
preservation desired (short or long term), and the facilities available. Short term and
long-term preservation techniques are employed routinely because bacterial strains
often loose desired properties or characteristics after repeated transfer of microbial
cells on culture media.
In this study other than preservation of strains at room temperature, cooling
(4ºC in the refrigerator) and freezing systems (-20ºC and -80ºC freezers) were
utilized. The cooling process in the cooling systems involved reduction of
temperatures of preservation solutions that resulted in temperature reduction hence
decreased molecular motions as do the rates of biological reactions as shown by
Arrhenius relationship of rates. The characteristic of the freezing system used in the
study has a coexistence of at least two phases the liquid phase and the solid phase
(Jay, 1996). In freezing systems, as the temperature is lowered there is a reduction in
mobility of the liquid phase. This leads to reduced biological activities, because of
heat transfer processes and thus the freezing interface of pure water or any other
medium is not zero (Corry, 1987). Presence of soluble solutes in water alters the
phase relationship between the ice and liquid (Mazur, 1970) since the process of
60
freezing involves loss of water from the cell through osmosis to the external
environment, a factor that influences the internal freezing process and freezing rate.
There is external ice formation while the cell contents are frozen, this osmotic transfer
increases the internal cell freezing point and hence the degree of supercooling.
From results in grading of growth on the LJ slopes after monthly subcultures
for five consecutive months, the MTB strains showed both intra-isolate and interisolate variation of cultural grading of growth when using the systematic approach.
The most effective temperature for preservation which yielded a consistent amount of
growth on Lowenstein Jensen medium after subcultures of strains in all suspending
media was -80°C and this was followed by -20°C. Survival could have been the
resultant of lowering of temperature and therefore reduction of biological activity,
including the biochemical reactions which lead to cell death.
The media of preservation also played a major role but could have been
affected by the preservation period and/or the manipulation process. Sterilized
distilled water as medium of preservation had lowest concordance at sub zero
temperatures. Discordant grading at room temperature could be due to continuity of
cellular activity even if not optimum in the non nutritious media leading to cell death.
Reduction of temperature could have played an important role to increased grading at
4Cº and at sub zero temperatures (-20ºC and -80ºC). At sub zero temperatures
reduced concordance in grading could be due to both water outflow and intracellular
ice crystal formation associated with cellular injury, since cells may not have been
limitlessly elastic to be able to shrink to a certain volume or expand without injury
61
with frequent thawing and freezing processes (Wolfe et al, 1985). Chances of
formation of intracellular ice or/and extra-cellular ice was high since water was used
as the major constituent in all media. This could have increased the chances of cells
being entrapped between ice crystals be subjected to mechanical and adhesion stress
that is detrimental to the survival of the cell (Grout et al, 1990). Reduction of
concordance in grading (fig 13) showed the unsuitability of using water (even for
short term) as a preservation medium for MTB.
Since a good protector should provide cryoprotection of cells during freezing
which provide a good matrix to allow stability (Costa et al., 2000) additives in water
played an important role in preservation of MTB strains. However there was varying
growth at the different temperatures used as shown by the level of concordance after
each month of preservation. At sub zero temperatures protective properties of
additives were enhanced than at temperatures above zero.
Lactose (15% aqueous solution of lactose at pH 5.0) as a medium of
preservation had better protective capability than sterilized distilled water. However,
solution effects due to the concentration of lactose during freezing could have
resulted exclusion of water from crystal structure causing an efflux water from the
cell resulting in precipitation of cytoplasmic solutes and components (Mazur, 1970)
mechanical stress to cellular components, and rupture of cell membranes due to ice
crystal formation (Mazur, 1977; Souzu, 1989) could have been detrimental to the
preserved strains.
62
With the solubility of lactose in water being 1 in 4.63 measured %w/v there
was a high probability for it to nucleate. Nucleation is required for freezing to initiate
(Nickerson and Moore, 1974) and lactose when used crystallized easily. As observed
in temperatures above zero and sub zero, even with prolonged vortexing a complete
homogeneous solution was not achieved from the crystallized sugars. This could
indicate a corresponding crystallization in the MTB cell which would have had an
adverse effect on the cell.
The addition of the cryoprotective agent glycerol to the preservation media
including sodium glutamate, OADC enriched Middlebrook 7H9 and trypticase soy
broth prior to addition of the cell suspension could have minimized the potentially
deteriorating effects of chemical reactions such as generation of heat during cooling
in MTB cells assuring a more uniform exposure and reducing potential toxic effects
of metabolism. The use of glycerol could have protected the cells from damage
during freezing and thawing processes (Bhat et al, 2005). It has been shown that if
vitrification solutions are not used, the cells being preserved are often damaged due to
freezing especially when they approach low temperatures or warming to room
temperature leading to low viable cell counts. Vitrification of glycerol could have
provided protection from damage due to intracellular ice formation since glycerol
directly diffuses through MTB lipid membranes in vitro (Paula et al., 1996).
Protection of the strains could be due to enzymatic digests of protein
substrates in trypticase soy broth which may have acted as protective colloids and
together with glycerol provided both intracellular and extracellular protection during
63
freezing (Gherna, 1994). However, at the room temperature this medium broth had no
protective effect to the cells.
OADC enriched Middlebrook 7H9 medium containing glycerol was a better
medium with higher recovery as compared to Middlebrook 7H9 when used solely as
preservation medium at all temperatures. Constituents of OADC could have
prevented oxidative damage by scavenging of free reactive oxygen radicals due to
presence of catalase in the solution in a way that it destroys toxic peroxides that may
be present in the medium especially at room temperature where biological reactions
could be on going even if not optimal, while Oleic acid could afford important fatty
acid containing carboxylic acid with a long unbranched aliphatic tail (chain)
important in MTB metabolism; Dextrose availed energy source; while albumin could
have acted as a protective agent by binding free fatty acids, which may be toxic to
MTB cells. The availability of these constituents could have ensured continuity of
metabolic activity at temperatures above zero and protection at sub zero temperatures.
Sodium glutamate with glycerol was an effective protectant for the MTB
strains at all the temperature used in all the months of preservation even with the
thawing and freezing processes for sub zero temperatures. The high recovery could
be due to ability of monosodium glutamate stabilizing protein structures of the
preserved strains by reactions between the amino groups of the protectant and the
carboxyl groups of the microorganism proteins. Although the mechanism of
protecting living cells by polyols is not fully understood, three hypotheses proposed
this include; maintenance of turgor resulting from the accumulation of mannitol at
64
low water activity, stabilization of the structures of membrane lipids and proteins at
low water activity and prevention of oxidative damage by scavenging of free reactive
oxygen radicals (Meister1979).
The stabilization of protein structures by reactions between the amino groups
of the protectant and the carboxyl groups of the microorganism proteins is another
protective mechanism of sodium glutamate. The presence of glycerol in the medium
could have had a proactive effect on the preservation suspension especially at sub
zero temperatures.
Gelatin medium had varying recovery at the temperatures of preservation but
high recovery at -80. The ability of gelatin to sustain viability at room temperature
could be due to its ability to form a strong structure that is quite chemically stable and
can even act as a buffer to reduce the affect of by products produced during cellular
metabolism as well as behaving as an acid or a base (amphoteric). As the pH shifted
from the basic towards a more acidic condition, the amino groups changed to become
positively charged, while a similar swing towards a more basic condition resulted in
changes in carboxylic group to become more negatively charged (Ward and Courts,
1977).
There was irregular concordance in Skimmed milk medium through the five
months of preservation when monthly readings were correlated with initial grading.
Variation of grading with skimmed milk in subsequent months could have resulted
from the color of the medium being a white solution. This had its own disadvantages
which included inability to establish a macroscopic visual homogeneity during
65
preparation of the preservation suspension as well as in the sub-culturing process.
There was also formation of a mushy coagulum causing a problem in producing a
standard inoculum during inoculation. This could have had a great impact since
mycobacteria clump. Also the medium dissociated into sediments at room
temperature and thus had little protective value as seen from the results. However
even with these drawbacks recovery after preservation with skimmed milk was good
at -80˚C (figure 6) this could be due to ability of Skimmed milk to prevent cellular
injury by stabilizing the cell membrane constituents (Valde´z et al. 1983; Kearney et
al. 1990; Castro et al. 1995)
Phosphate buffered saline which is normally isotonic and non-toxic to cells is
known to structure water around biomolecules and such thin film of water prevents
denaturing of biomolecules or conformational changes to cells, a factor that could
have been utilized in the first two months at room temperature. On the other hand this
additive could have protected the strains from the effects of thawing and freezing
process but the storage temperature played a key role in preservation as seen with
high recovery at sub zero temperatures and especially at -80ºC.
The cell wall structure may have contributed enormously to the survival of
MTB strains at different temperatures and media due to presence of an extremely
hydrophobic cell wall forming an exceptionally strong permeability barrier as
ascribed to the unique structure containing long chain fatty acids (C60 to C90); the
mycolic acids (Minnikin, 1982) as well as having 100 to 1000 fold lower channel
forming proteins (porins) than for E. coli (Jarlier and Nikaido, 1990). The membrane
66
damage being more detrimental than cell wall damage (Mazur 1965, 1977), presence
of intracellular ice crystals could have interfered with the plasma membrane due to its
low permeability as well as thawing and freezing rate both on the inside and outside
cellular environment.
Even in sterilized distilled water some cells were viable even at room
temperature and at 4ºC (fig 16 and 17 respectively) for five months. The cell structure
and physiology could have played a major role. Trehalose a disaccharide is the major
free sugar in the cytoplasm of mycobacteria; it is a constituent of cell wall
glycolipids, and it plays a role in mycolic acid transport during cell wall biogenesis
(Murphy et al., 2005). It has a protective effect on proteins and biological membranes
during cryopreservation or desiccation in vitro, and has been implicated in survival of
micro-organisms exposed to environmental stresses in vivo (Koen et al., 2000)
The phase behavior of mycolate containing lipid, trehalose dimycolate (cord
factor) in mycobacteria forms a presumably para-crystalline structure (Durand et al
1979). How trehalose provides protection to cells is not entirely clear, both in vivo
and in vitro evidence has been obtained for dual mechanism: stabilization of
membranes and proteins by replacing water and preservation of intracellular water
structure (Clegg, 1985; Burke, 1985; singer and Lindquist, 1998; Sano et al, 1999).
The cell wall structure may act as a sheath which prevents inoculative freezing and
allows MTB cells to supercool in the presence of external ice.
Damage of the cells associated with changes occurring either during freezing
or thawing process are associated with ice formation either directly (mechanical
67
effect) or indirectly or changes in solid concentration in a unfrozen phase, migration
of water from cell interior to cell exterior produce cell shrinkage and membrane
damage through phase transformation in non-aqueous membrane components
(liquids). Time of exposure to high solute concentration during freezing and thawing
process can cause cell damage. In many systems, reaction rates as a function of
temperature go through a maximum at some temperature below the initial freezing
point. The moisture migration within the cell occurs through the freezing process.
Supercooling of the cell content can lead to moisture movement through an osmotic
mechanism.
Thawing causes internally frozen cells to rehydrate very fast and the medium
surrounding the microbial cells is diluted due to the phase shift and cell are exposed
to osmotic shock there by causing cell death (Calcott and Thomas, 1978). Survival of
MTB strains to the thawing and freezing process as observed could be due to cell wall
structure that allows limited movement of hydrophilic molecules. The repeat of the
subculture exercise using approach two could be a contributing factor to reduction in
grading since there was a repeat in freezing and thawing. This could have allowed
selective influx and efflux of liquids in the cells resulting to membrane damage since
the probability intracellular ice formation was high.
Different types of cells may require different cooling rates; a uniform cooling
rate of 1°C per minute from ambient temperature is effective for a wide variety of
cells. Fast freezing produces internal freezing in cells causing cell death while slow
freezing produces cellular dehydration and only extra-cellular ice. This phenomenon
68
could explain why the strains had reduced grading in growth since the change was
almost drastic thus from room temperature to 4ºC to -20ºC then to -80ºC and not
consistent cooling by lowering degree by degree.
Clumping of the cells was high at 4ºC temperature and at room temperature
even in the freshly homogenized suspensions. The high tendency of M. tuberculosis
strains to clump could be due to the high lipid content on their cell wall structure. At
sub zero temperatures clumping could have occurred in the liquid phase before
freezing and more during freezing due to selective selection of substances contained
in the preservation suspension. At these temperatures there is nucleation which causes
solutes to crystallize and cells to clump together. This could account for differences in
grading in subsequent months where by a low grade could precede a high grade as
seen in figures (6, 7, and 8).
Results obtained from the cross tabulation of each preservation medium
systematically thawed with the corresponding medium thawed directly (figure 19)
from
-80ºC through to room temperature sterilized distilled water was highly
affected by the thawing process while OADC enriched 7H9, skimmed milk,
trypticase soy broth and gelatin medium were least affected even though there was no
significant difference in the between the two recovery processes. Presence of
cryopreservation agent glycerol could have had a significant role in protecting the
cells from the direct change in temperatures from -80ºC. In OADC enriched 7H9 and
trypticase soy broth by preventing the direct drastic temperature change. While high
concordance with both approaches in preservation and recovery by skimmed milk
69
could be due to prevention of cellular injury by stabilization of the cell membrane
constituents (Valde´z et al. 1983; Kearney et al. 1990; Castro et al. 1995)
Although the different preservation medium had different abilities to sustain
viability of the strains it is important to note that within one month of preservation all
the medium used had high concordance grading readings and thus even at room
temperature the viability of the MTB strains could be maintained. This is important
especially in the resource limited countries which would require shipment of strains
for further analysis or for external quality control and proficiency testing by the
supranational laboratories.
70
CHAPTER SIX
6.3CONCLUSIONS
Although tuberculosis has been of health concern for a long period of time,
preservation of the isolated strains by culture has received little attention. In this
study, it has been observed that recovery of M. tuberculosis strains after five months
cryopreservation and monthly subcultures depends on the temperature of preservation
and medium used. The recovery of M. tuberculosis after preservation is optimum at
sub zero temperatures and specifically at -80˚C.
Together with temperature it is evident that M. tuberculosis requires
suspension media additives for optimum recovery especially at sub zero temperatures.
Different media have different capabilities when preserving M. tuberculosis strains at
the respective temperature utilized. The media which was optimum in preservation of
M. tuberculosis strains in all the temperatures utilized was Sodium glutamate 5% with
glycerol. However, M. tuberculosis strains even without additives could survive in
water when used as the suspending media for five months at -80˚C, -20˚C, 4˚C and
room temperature.
Mycobacteria tuberculosis can be preserved by either direct thawing or
systematic thawing since there is no significant effect on growth recovery for strains
when stored at -80˚C temperature. However, monthly repeats of systematic approach
method in preservation and recovery of M. tuberculosis resulted in lower recovery
than does in direct approach preservation and recovery.
71
6.4RECOMMENDATIONS
With the demonstration that there is reduction in recovery of M. tuberculosis
when systematic approach is used in monthly subculture process, than direct approach
when used in freezing and recovery after five months. It is therefore important to
determine effects of using approach one and recovery using approach two and vice
versa in preservation and recovery of M. tuberculosis strains.
In order to established a conclusive standardized system that will achieve high
recovery of M. tuberculosis it will be essential to determine the effects of temperature
and medium in preservation of M. tuberculosis when archived for more than five
months.
Since there was monthly thawing and freezing when using the systematic approach it
would be important to determine if there is loss of virulence when preserving at sub
zero temperatures or above zero temperatures.
72
7.0
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83
APPENDIX1
SOPs for Media used for preservation
A. OADC enriched Middlebrook 7H9 Broth + 5% glycerol
1. Suspend 4.7 g of the dehydrated powder in 850 mL of purified water 2.
2. Add 50ml glycerol.
3. Autoclave at 121°C for 10 min.
4. Aseptically add 100 ml of Middlebrook OADC Enrichment to the medium when
cooled to room temperature and dispense directly 1ml into 2ml cryovials.
5. Quality control was performed on the prepared medium
B. Preparation of Middlebrook 7H9 Broth (BD) (Cat. Number 296068)
1. Dissolve 4.7 g of Middlebrook 7H9 Broth powder was in 1000 ml of sterilized
distilled water.
2. Autoclave the mixture at 121˚C for 15 min.
3. Dispense the medium into 20ml universal glass bottles.
4. Quality control was done on the prepared medium.
3.
Trypticase™ Soy Broth (BD)
Formula
Trypticase Soy Broth Formula Per Liter
Pancreatic Digest of Casein ...................................... 17.0 g
Enzymatic Digest of Soybean Meal ............................ 3.0 g
Sodium Chloride ........................................................ 5.0 g
Dipotassium Phosphate .............................................. 2.5 g
84
Dextrose ..................................................................... 2.5 g
Method of Preparation Trypticase™ Soy Broth 20% Glycerol
1. Suspended 27.5 -grams Tryptic Soy Broth w/o Dextrose medium in 800ml
distilled or deionized water in Erlenmeyer flasks.
2. Add 200ml glycerol to the 1000ml mark.
3. Heat with gentle agitation to dissolve. Final pH, 7.3 ± 0.2.
4. Dispensed in 20ml universal glass bottles.
5. Autoclaved at 121°C for 15 min.
6. Cool to room temperature and stored at 4˚C.
C. Skim Milk Medium Difco™ (Cat. No. 232100)
Directions for Preparation from dehydrated Product
1. Dissolve 100 g of the powder in 1 L of sterilized distilled water.
2. Warm to completely dissolve the powder.
3. Autoclave at 121˚C for 15 min.
6. Quality control was performed on the prepared medium
D. BBL™ Lactose Broth
Approximate Formula* Per Liter
Cat. Number 221893 Becton and Dickson
Beef Extract
3.0 g
Pancreatic Digest of Gelatin
5.0 g
Lactose
5.0 g
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Method
1. Dissolve 150g of the powder in 1 L of sterilized distilled water.
2. Dispense in test universal glass bottles, in 20 ml amounts
3. Autoclave at 121°C for 10 min at 10 pounds pressure or Tyndallization to reduce
the hydrolysis of lactose.
4. Quality control was performed on the prepared medium.
E. Preparation of Phosphate Buffered Saline
BBL™ Phosphate Buffer, pH 7.2
Approximate Formula* Per Liter
Potassium Dihydrogen Phosphate
26.22 g
Sodium Carbonate
7.78 g
Directions for Preparation from dehydrated Product
1. Prepare a stock solution, dissolving 34.0 g in purified water and make up to 1 L.
2. Prepare a working solution by adding 1.25 ml of stock phosphate buffer solution
to sterilized distilled water and make up to 1 L (1:800).
3. Dispense into 20ml universal bottles and autoclave at 121°C for 15 min, store
under refrigeration. (Note: pH may vary depending on glassware used and may
require additional adjustment to achieve 7.2 )
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F. Preparation of Sodium glutamate medium
Sodium glutamate Difco™
5.0g
Glycerol
6ml
Distilled water
100ml
1. Dissolve the ingredients in distilled water by heating.
2. Dispense into 20ml universal bottles and autoclave at 121°C for 30 min, store
under refrigeration.
3. Quality control was performed on the prepared medium.
G. Preparation of media for subculture of MTB strains
Preparation of Lowenstein Jensen- glycerol medium
Requirements include the preparation of:
1. Mineral salts
2. Homogenized whole Egg base
3. Malachite green solution
4. Penicillin solution (made by dissolving 1,000,000 IU penicillin in 2ml sterile
distilled water and making up the volume to 10 ml. final conc. 100,000 IU)
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Table 1: Preparation of mineral salts
Reagent
Amount
Potassium dihydrogen phosphate anhydrous (KH2PO4)
4g
Magnesium sulphate (MgSO4. 7H2O)
0.4g
Magnesium citrate -
1g
L-Asparagine
6g
Glycerol (reagent grade)
20ml
Distilled water
1000ml
Procedure
1. Weigh the Salts as indicated above and transfer into sterile 2000ml volumetric
flask
2.
Dissolve completely in distilled water by heating with occasional swirling.
3.
Sterilize by autoclave the g solution at 1210C for 30 min.
4.
Cool to room temperature and store in a 40 C refrigerator at.
Malachite green solution 2%
Malachite green dye
2.0g
Sterile distilled water
100ml
Dissolve malachite green dye completely in distilled water and aliquot in 20mls
volumes into universal bottles then autoclave at 1210C for 30 min.
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Preservation
Give a batch number to the LJ medium in the media log book and store at 4 -80 C
ready for use up to 4 weeks.
Preparation of egg base
1. Wipe all the benches with a cotton swab soaked in 95% methyl alcohol.
2. Soak fresh eggs (up to 3 days old) for 5 min in plain devo clean solution, clean by
gently scrubbing with a hand brush and thoroughly rinse in running tap water and
then allowed to dry.
3. Wipe the eggs with cotton wool soaked with methylated spirit and break them
into a graduated glass jar then pour into a sterile 6 L volumetric flask with sterile
3mm glass beads to make 500ml.
4. Shake the contents vigorously to homogenize the mixture.
5. Aseptically add 20ml malachite green solution, mineral salt solution 600ml and
shake well before sieving the mixture and adding penicillin drug in the ratio of
1ml: 1000ml of egg based media and shake well.
6. Dispense 5 ml amounts into universal bottles and inspissate at 850C for 1 hour.
89
APENDIX3
Table 2:
Reporting of subculture results
Reading
Report
Grading
No Growth
Negative
NGO
1 – 19 colonies
Positive
Number of colonies
20-100 colonies
Positive
1+
>100 colonies
Positive
2+
Confluent growth
Positive
3+
Contaminated
Contaminated
*
World health organization culture grading system for M. tuberculosis
90
APENDIX4
Quality control
Check for growth support
Before use the following procedures were used as quality control to test for the
aptness of each media.
Lowenstein Jensen media
Lowenstein Jensen media was tested by inoculation of Mycobacterium tuberculosis
strain H37RV. The slopes were incubated at 37°C and examined weekly for growth
or contamination up to four week after which if no growth was observed the slopes
were regarded as non optimal, however if positive the media was regarded as
optimum.
Un-inoculated media were used as negative control as well as to check for
contamination of the media.
OADC Middlebrook 7H9 Broth + Glycerol
After preparation of enriched OADC Middlebrook 7H9 Broth + Glycerol the media
was tested for performance characteristics. Using a 0.01 ml calibrated loop colonies
of M. tuberculosis H37RV were inoculated in a representative sample of 4ml of the
product. The tubes were incubated at 37°C and tubes were examined for growth after
7, 14 and 21 days. Turbidity in less than five days of inoculation and incubated was
regarded as contamination.
Skimmed milk
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The prepared skimmed milk was tested for performance characteristics by inoculating
a loopful of fresh culture of Escherichia coli ATCC 25922 into a tube containing
freshly prepared skimmed milk media. The media wasthen incubated at 35°C for 7
days. The media was regarded as adequate when there was formation of acid and
curdling.
Lactose
The prepared Lactose was tested for performance by inoculating fresh colonies of
Neisseria lactamica ATCC 23970 into the prepared media containing a pH indicator.
The tubes were incubated at 35°C in an aerobic atmosphere and examined after 4 h.
Color change from red to yellow is indicated support of growth.
Sodium Glutamate
Sodium Glutamate media was tested for performance by inoculating Enterobacter
aerogenes ATCC 13048 into the freshly prepared media and incubating at 35°C for
18-48 hours for acid and gas production.
92